Triacylglycerol (TAG) is thought to be the most important storage of energy for cells. Diacylglycerol acyl transferase is an enzyme which is believed to regulate TAG structure and direct TAG synthesis. The reaction catalyzed by DAGAT is at a critical branchpoint in glycerolipid biosynthesis. Enzymes at such branchpoints are considered prime candidates for sites of metabolic regulation. There are several enzymes which are common to the synthesis of diacylglycerol, TAG and membrane lipids, however, the DAGAT reaction is specific for oil synthesis.
In plants, TAG is the primary component of vegetable oil that is used by the seed as a stored form of energy to be used during seed germination. Higher plants appear to synthesize oils via a common metabolic pathway. Fatty acids are made in plastids from acetyl-CoA through a series of reactions catalyzed by enzymes known collectively as Fatty Acid Synthetase (FAS). The fatty acids produced in plastids are exported to the cytosolic compartment of the cell, and are esterified to coenzyme A. These acyl-CoAs are the substrates for glycerolipid synthesis in the endoplasmic reticulum (ER). Glycerolipid synthesis itself is a series of reactions leading first to phosphatidic acid (PA) and diacylglycerol (DAG). Either of these metabolic intermediates may be directed to membrane phospholipids such as phosphatidylglycerol (PG), phosphatidylethanolamine (PE) or phosphatidylcholine (PC), or they may be directed on to form neutral triacylglycerol (TAG).
Diacylglycerol (DAG) is synthesized from glycerol-3-phosphate and fatty acyl-CoAs in two steps catalyzed sequentially by glycerol-3-phosphate acyltransferase (G3PAT), and lysophosphatidic acid acyltransferase (LPAAT) to make PA, and then an additional hydrolytic step catalyzed by phosphatidic acid phosphatase (PAP) to make DAG. In most cells, DAG is used to make membrane phospholipids, the first step being the synthesis of PC catalyzed by CTP-phosphocholine cytidylyltransferase. In cells producing storage oils, DAG is acylated with a third fatty acid in a reaction catalyzed by diacylglycerol acyltransferase (DAGAT). Collectively, the reactions make up part of what is commonly referred to as the Kennedy Pathway.
Diacylglycerol acyltransferase (hereinafter referred to as DAGAT or DGAT) is an integral membrane protein that catalyzes the final enzymatic step in the production of triacylglycerols in plants, fungi and mammals. DGAT has generally been described in Harwood, J. Biochem. Biophysics. Acta, 1301:7-56 (1996); Daum G., et al. Yeast 16:1471-1510 (1998); and Coleman, R., et al. Annu. Rev. Nutr. 20:77-103 (2000) (all of which are herein incorporated by reference). This enzyme is responsible for transferring an acyl group from acyl-coenzyme-A to the sn-3 position of 1,2-diacylglycerol (DAG) to form triacylglycerol (TAG). As the final step in TAG biosynthesis via the Kennedy pathway, it is the only step not involved in membrane biosynthesis. In plants and fungi DGAT is associated with the membrane and lipid body fractions, particularly in oilseeds, where it contributes to the storage of carbon used as energy reserves. In animals, the role of DGAT is more complex. Triacylglycerols are synthesized and stored in several cell types including adipocytes and hepatocytes (Bell, R. M., et al. Annu. Rev. Biochem. 49:459-487 (1980) (herein incorporated by reference)) but in addition, DGAT may play a role in lipoprotein assembly and the regulation of plasma triacylglycerol concentration (Bell, R. M., et al.), as well as participate in the regulation of DAG levels (Brindley, D. N. Biochemistry of Lipids, Lipoproteins and Membranes, eds. Vance, D. E. & Vance, J. E. (Elsevier, Amsterdam), 171-203; and Nishizuka, Y. Science 258:607-614 (1992) (both of which are herein incorporated by reference)).
The structure of the TAG, as far as positional specificity of fatty acids, is determined by the specificity of each of the three acyltransferases for the fatty acyl-CoA and the glycerol backbone substrates. Thus, for example, there is a tendency for the acyltransferases from many temperate zone species of seeds to allow either a saturated or an unsaturated fatty acid at the sn-1 or the sn-3 position, but only an unsaturated fatty acid at the sn-2. The absolute specificity for an unsaturated fatty acid at sn-2 is determined by the substrate preference of LPAAT enzyme. In some species such as cocoa, TAG compositions suggest that this tendency is carried further in that there is an apparent preference for acylation of the sn-3 position with a saturated fatty acid, if the sn-1 position is esterified to a saturated fatty acid. Thus, there is a higher percentage of structured TAG of the form SUS (where S=saturated fatty acid and U=unsaturated fatty acid), than would be expected from a random distribution based on the overall fatty acid composition with the sn-2 position fixed with an unsaturated fatty acid. This suggests that DAGAT also plays an important role in the regulation of TAG structure, if not also in the control of TAG synthesis.
Obtaining nucleic acid sequences capable of producing a phenotypic result in the incorporation of fatty acids into a glycerol backbone to produce an oil is subject to various obstacles including but not limited to the identification of metabolic factors of interest, choice and characterization of a protein source with useful kinetic properties, purification of the protein of interest to a level which will allow for its amino acid sequencing, utilizing amino acid sequence data to obtain a nucleic acid sequence capable of use as a probe to retrieve the desired DNA sequence, and the preparation of constructs, transformation and analysis of the resulting plants.
Thus, the identification of enzyme targets and useful tissue sources for nucleic acid sequences of such enzyme targets capable of modifying oil structure and quantity are needed. Ideally an enzyme target will be amenable to one or more applications alone or in combination with other nucleic acid sequences relating to increased/decreased oil production, TAG structure, the ratio of saturated to unsaturated fatty acids in the fatty acid pool, and/or to other novel oils compositions as a result of the modifications to the fatty acid pool.
For example, in some instances having an oilseed with a higher ratio of oil to seed meal would be useful to obtain a desired oil at lower cost. This would be typical of a high value oil product. Or such an oilseed might constitute a superior feed for animals. In some instances having an oilseed with a lower ratio of oil to seed meal would be useful to lower caloric content. In other uses, edible plant oils with a higher percentage of unsaturated fatty acids are desired for cardiovascular health reasons. And alternatively, temperate substitutes for high saturate tropical oils such as palm, coconut, or cocoa would also find uses in a variety of industrial and food applications.
In mammals, DAGAT plays an important role in the metabolism of cellular diacylglycerol and is important in processes involving triacylglycerol metabolism including intestinal fat absorption, lipoprotein assembly, adipose tissue formation and lactation. As such, identification and isolation of the DAGAT protein and of polynucleotide and polypeptide sequences is desired.
Several putative isolation procedures have been published for DAGAT. Polokoff and Bell (1980) reported solubilization and partial purification of DAGAT from rat liver microsomes. This preparation was insufficiently pure to identify a specific protein factor responsible for the activity. Kwanyuen and Wilson (1986, 1990) reported purification and characterization of the enzyme from soybean cotyledons. However, the molecular mass (1843 kDa) suggests that this preparation was not extensively solubilized and any DAGAT protein contained therein was part of a large aggregate of many proteins. Little et al (1993) reported solubilization of DAGAT from microspore-derived embryos from rapeseed, but as with Kwanyuen and Wilson, the molecular mass of the material that was associated with activity was so high, that complete solubilization is unlikely. Andersson et al (1994) reported solubilization and a 415-fold purification of DAGAT from rat liver using immunoaffinity chromatography. However, there is no evidence that the antibodies they used recognize DAGAT epitopes, nor that the protein that they purified is truly DAGAT. Indeed, as with Kwanyuen and Wilson, the DAGAT activity in their preparations exhibited a molecular mass typical of aggregated membrane proteins. Finally, Kamisaka et al (1993, 1994, 1996, 1997) report solubilization of DAGAT from Mortierella rammaniana and subsequent purification to homogeneity. They suggest that DAGAT solubilized from this fungal species has an apparent molecular mass of 53 kDa by SDS-PAGE. However, as shown in Example 4 below, fractions obtained using the protocol described by Kamisaka et al. did not provide abundant 53-kDa polypeptide which correlated with DAGAT activity.
Cases et al. reported a cloning of a DGAT gene from mouse. Using coding sequences from acyl CoA:cholesterol acyltransferase (ACAT), EST databases were searched and a gene identified that shared 20% identity with the mouse ACAT. After cloning and expression of the gene in insect cells no ACAT activity was reported in isolated membranes. Using [1-14C]oleoyl-CoA as substrate a range of acceptors was examined and Cases et al. reported DAG as the acceptor molecule. Hobbs et al. (1999) FEBS Letters 452:145-149 (herein incorporated by reference) reported the cloning of an Arabidopsis homologue of the mouse DGAT gene and reported the presence of DGAT activity in insect cells expressing the cDNA. Southern analysis indicated a single gene copy was present in Arabidopsis. Katavic et al. (1995) Plant Physiol. 108:399-409 and Zou et al. (1999) The Plant Journal 19:645-653 (both of which are herein incorporated by reference) also reported this gene in seed oil production when an insertional mutation (AS11) in the gene was found to lower seed oil levels and decrease DGAT activity. The locus, at approximately 35 cM on chromosome II, was designated TAG1. Routaboul J. M., et al. (1999) Plant Physiol. Biochem. 37:831-840 (herein incorporated by reference) reported similar results identifying an Arabidopsis mutant (ABX45) harboring a frame-shift mutation near the 5′ end of the TAG1 reading frame. This mutation resulted in a complete change in coding sequence after the first 60 amino acids. With the identification of a single DGAT gene copy in Arabidopsis and the detection of DGAT activity even after a frame shift mutation disabled gene translation, Routaboul et al. concluded that another protein must be responsible for the remaining DGAT activity.